Passage
A newborn girl is diagnosed with a rare genetic disorder that causes premature lysis of red blood cells. Through genome sequencing, physicians identified a specific mutation (R510Q) in both copies of her pyruvate kinase gene, which encodes the enzyme that catalyzes the final step of glycolysis. Pyruvate kinase produces pyruvate and ATP from phosphoenolpyruvate (PEP) and ADP.PEP + ADP ↔ Pyruvate + ATPReaction 1Because red blood cells lack mitochondria, pyruvate in erythrocytes does not enter the citric acid cycle and the electron transport chain. Therefore, red blood cells rely almost exclusively on glycolysis to produce sufficient ATP for cellular functions. In patients with pyruvate kinase deficiency, it is thought that a lack of ATP compromises the integrity of the red blood cell membrane, leading to premature lysis in the spleen. Although these observations are well established, the exact mechanism by which low ATP alters the cell membrane is unclear.Scientists interested in analyzing this particular patient's gene variant expressed and purified recombinant R510Q pyruvate kinase. They performed several biochemical tests to characterize the R510Q mutant with respect to wild-type (WT) pyruvate kinase. In the first experiment, WT and R510Q pyruvate kinases were each incubated with a constant amount of ADP and varying amounts of PEP. The activity of the enzyme was measured at each concentration of PEP and plotted. They then repeated the experiment in the presence of ATP and found that ATP is a noncompetitive inhibitor of the WT enzyme, but a mixed inhibitor of the R510Q enzyme. The results are shown in Figure 1. All experiments were done at the same enzyme concentration.
Figure 1 Pyruvate kinase activity with and without ATP as a function of [PEP]. Open markers are shown at ½V_max for each plot.
Adapted from Wang C, Chiarelli LR, Bianchi P, et al. Human erythrocyte pyruvate kinase: characterization of the recombinant enzyme and a mutant form (R510Q) causing nonspherocytic hemolytic anemia. Blood. 2001;98(10) :3113-20.
-Based on Figure 1, which of the following statements is true regarding R510Q pyruvate kinase as compared to WT pyruvate kinase?

Question

Passage
A newborn girl is diagnosed with a rare genetic disorder that causes premature lysis of red blood cells. Through genome sequencing, physicians identified a specific mutation (R510Q) in both copies of her pyruvate kinase gene, which encodes the enzyme that catalyzes the final step of glycolysis. Pyruvate kinase produces pyruvate and ATP from phosphoenolpyruvate (PEP) and ADP.PEP + ADP ↔ Pyruvate + ATPReaction 1Because red blood cells lack mitochondria, pyruvate in erythrocytes does not enter the citric acid cycle and the electron transport chain. Therefore, red blood cells rely almost exclusively on glycolysis to produce sufficient ATP for cellular functions. In patients with pyruvate kinase deficiency, it is thought that a lack of ATP compromises the integrity of the red blood cell membrane, leading to premature lysis in the spleen. Although these observations are well established, the exact mechanism by which low ATP alters the cell membrane is unclear.Scientists interested in analyzing this particular patient's gene variant expressed and purified recombinant R510Q pyruvate kinase. They performed several biochemical tests to characterize the R510Q mutant with respect to wild-type (WT) pyruvate kinase. In the first experiment, WT and R510Q pyruvate kinases were each incubated with a constant amount of ADP and varying amounts of PEP. The activity of the enzyme was measured at each concentration of PEP and plotted. They then repeated the experiment in the presence of ATP and found that ATP is a noncompetitive inhibitor of the WT enzyme, but a mixed inhibitor of the R510Q enzyme. The results are shown in Figure 1. All experiments were done at the same enzyme concentration.

Figure 1 Pyruvate kinase activity with and without ATP as a function of [PEP]. Open markers are shown at ½V_max for each plot.
Adapted from Wang C, Chiarelli LR, Bianchi P, et al. Human erythrocyte pyruvate kinase: characterization of the recombinant enzyme and a mutant form (R510Q) causing nonspherocytic hemolytic anemia. Blood. 2001;98(10) :3113-20.
-Based on Figure 1, which of the following statements is true regarding R510Q pyruvate kinase as compared to WT pyruvate kinase?

Quizplus · Accepted Answer

11ecba2d_0e4e_747c_a3bf_7bb35a46536e_MD0008_00 Enzyme kinetics are frequently described using the Michaelis-Menten equation, which calculates the reaction velocity at varying substrate concentrations. On a graph of reaction velocity V_o versus substrate concentration [S], the maximum velocity V_max is found at the plateau of the curve. V_max represents the reaction rate when an enzyme's active sites are completely saturated with substrate, and it is directly proportional to the total enzyme concentration and the turnover number k_cat. The Michaelis constant K_m is the substrate concentration at which ½V_max is achieved. Therefore, K_m values can be found by identifying the substrate concentration at which the curve reaches half of its plateau height. Figure 1 shows reaction velocity versus [S] plots for WT and R510Q pyruvate kinase in the presence and absence of ATP. In the absence of ATP, WT and R510Q pyruvate kinase behave almost identically. Both enzymes have about the same V_max and K_m values because their curves plateau at the same height and reach half the maximum height at approximately the same substrate concentration. Because the reactions are run at equal enzyme concentrations, the similar V_max values also indicate similar k_cat values. However, in the presence of ATP, WT pyruvate kinase has a higher V_max (and k_cat) and a lower K_m than R510Q pyruvate kinase. (Choices A and B) In the presence of ATP, the K_m of R510Q pyruvate kinase increases rather than decreasing or remaining the same. (Choice D) When ATP is added, the V_max (and therefore the k_cat) of R510Q pyruvate kinase is much lower than that of WT pyruvate kinase. Educational objective: Enzyme activity is expressed mathematically by the Michaelis-Menten equation, 11ecba2d_0e4e_9b8d_a3bf_e78b0aff5079_MD0008_00 Vo=VmaxS Km + S. V_max is directly proportional to k_cat and enzyme concentration, and it is indicated by the height of the plateau on a Michaelis-Menten graph. The Michaelis constant K_m is the substrate concentration at which ½V_max is achieved.

Passage A Newborn Girl Is Diagnosed with a Rare Genetic Disorder